title: A mechanistic study of Trichoderma reesei Cel7B catalyzed glycosidic bond cleavage.
abstract: An ONIOM study is performed to illustrate the mechanism of Trichoderma reesei Cel7B catalyzed p-nitrophenyl lactoside hydrolysis. In both the glycosylation and deglycosylation steps, the reaction proceeds in a concerted way, meaning the nucleophilic attack and the glycosidic bond cleavage occur simultaneously. The glycosylation step is rate limiting with a barrier of 18.9 kcal/mol, comparable to the experimental value derived from the kcat measured in this work. The function of four residues R108, Y146, Y170, and D172, which form a hydrogen-bond network involving the substrate, is studied by conservative mutations. The mutants, including R108K, Y146F, Y170F, and D172N, decrease the enzyme activity by about 150-8000-fold. Molecular dynamics simulations show that the mutations disrupt the hydrogen-bond network, cause the substrate to deviate from active binding and hinder either the proton transfer from E201 to O4(+1) or the nucleophilic attack from E196 to C1(-1).
authors.affiliation: Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266061, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Shihai
authors.initials: S
authors.lastname: Yan
authors.forename: Lishan
authors.initials: L
authors.lastname: Yao
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000494:pharmacology; D006027:Glycosides / Q000737:chemistry; D006860:Hydrogen Bonding; D006868:Hydrolysis; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D006027:Glycosides; C407024:endoglucanase Cel5A; D002480:Cellulase